Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.
GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.
Sleeman
M W
MW
Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA.
Donegan
N P
NP
Heller-Harrison
R
R
Lane
W S
WS
Czech
M P
MP
D000214:Acyl Coenzyme A / Q000378:metabolism; D017667:Adipocytes / Q000201:enzymology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D008297:Male; D008561:Membrane Fusion; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000214:Acyl Coenzyme A; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C495694:Slc2a4 protein, rat; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase